PURPOSE. To assess the corneal iontophoretic delivery of gentamicin by drug-loaded hydrogel probe, and to determine the resultant ocular disposition and elimination of the drug from the cornea and anterior chamber. METHODS. Corneal iontophoresis of gentamicin sulfate was studied in healthy white rabbits by using drug-loaded disposable hydroxyethyl methacrylate (HEMA) hydrogel disk probes and a portable mini-ion device designed in the authors' laboratory. The iontophoretic treatment was performed with a current intensity of 1 mA for 60 seconds only. Three control groups were used: mock iontophoresis (no current) for 60 seconds, topical eye drops of fortified gentamicin (1.4%) every 5 minutes for 1 hour, and subconjunctival injection of 0.25 mL of 40 mg/mL gentamicin solution. The animals in the iontophoretic experimental groups were killed at predetermined time points. The gentamicin concentrations in the cornea and aqueous humor were assayed with a fluorescence polarization immunoassay. Analysis of the gentamicin eye pharmacokinetics was performed with a modeling approach. RESULTS. Peak gentamicin concentrations in the cornea (363.1 ± 127.3 μg/g) and in the aqueous humor (29.4 ± 17.4 μg/mL) were reached at 0 and 2 hours after the iontophoretic treatment, respectively. The peak gentamicin concentrations after a single iontophoresis treatment were 12 to 15 times higher than those obtained after gentamicin injection or after topical eye drop instillation, and much higher than in mock iontophoresis. The concentration versus time profile of gentamicin in the cornea and the anterior chamber after iontophoresis was appropriately described by applying a two-compartment pharmacokinetic model. CONCLUSIONS. A short iontophoretic treatment using gentamicin-loaded hydrogels has potential clinical value in increasing drug penetration to the anterior segments of the eye and maintaining therapeutic drug levels in the cornea for more than 8 hours.